WO2005019811A2 - Mesures de fluorescence dependant du temps - Google Patents

Mesures de fluorescence dependant du temps Download PDF

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Publication number
WO2005019811A2
WO2005019811A2 PCT/US2004/027890 US2004027890W WO2005019811A2 WO 2005019811 A2 WO2005019811 A2 WO 2005019811A2 US 2004027890 W US2004027890 W US 2004027890W WO 2005019811 A2 WO2005019811 A2 WO 2005019811A2
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WO
WIPO (PCT)
Prior art keywords
light
sample
collection
optical elements
scanning
Prior art date
Application number
PCT/US2004/027890
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English (en)
Other versions
WO2005019811A3 (fr
Inventor
Evan F. Cromwell
Johann F. Adam
Andrei Brunfeld
Paul B. Comita
Christopher J. Seipert
Original Assignee
Blueshift Biotechnologies, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Blueshift Biotechnologies, Inc. filed Critical Blueshift Biotechnologies, Inc.
Priority to EP04786593A priority Critical patent/EP1660870A2/fr
Priority to JP2006524881A priority patent/JP2007504445A/ja
Publication of WO2005019811A2 publication Critical patent/WO2005019811A2/fr
Publication of WO2005019811A3 publication Critical patent/WO2005019811A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6445—Measuring fluorescence polarisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G02B21/0048—Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0068—Optical details of the image generation arrangements using polarisation
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6484—Optical fibres

Definitions

  • a sample such as a biological specimen is stained with fluorophores before being illuminated by light of a relatively short wavelength.
  • the illumination light which typically is provided from a laser, excites the fluorophores into a higher energy state where they remain for a short period of time, before returning to their original energy state while emitting fluorescent light of a wavelength longer than the excitation wavelength.
  • the emitted fluorescent light is collected by an objective lens of the microscope and is passed through the optical system of the microscope, such that it can be viewed by a user, for example, through the eyepieces of the microscope, or on a display screen of a video system that is connected to the microscope's optical system.
  • both the excitation light and the fluorescent light share an optical path through the microscope's optical system, and can be separated as needed, by optical components such as dichroic mirrors that reflect light above the excitation wavelengths while passing the excitation light.
  • optical components such as dichroic mirrors that reflect light above the excitation wavelengths while passing the excitation light.
  • the excitation light source can illuminate a portion of an object to be examined, such as one microlocation in an array of micro locations.
  • an object to be examined such as one microlocation in an array of micro locations.
  • 2P two-photon
  • the pulsed laser allows the same fluorophores to be excited by photons of twice the wavelength than those used in single photon systems, but the longer wavelength photons are not absorbed by the biological sample, which results in decreased toxicity to living cells and decreased photo bleaching.
  • the infrared wavelength excitation significantly reduces scattering within the tissue, as the scattering coefficient is proportional to the inverse fourth power of the excitation wavelength, resulting in penetration deeper into the specimen.
  • the invention provides methods and apparatus, including computer program products, implementing and using techniques for collecting optical data pertaining to one or more characteristics of a sample.
  • the apparatus has a light source, one or more illumination optical elements, a scanner, one or more collection optical elements, and a device forming an aperture that limits detection of light from the sample.
  • the illumination optical elements direct a light beam from the light source onto the sample.
  • the scanner scans the light beam across the sample.
  • the one or more collection optical elements collect light from the sample and transmit the collected light to a detector. None of the one or more collection optical elements are included among the one or more illumination optical elements.
  • the device forming an aperture limits detection of light from the sample to light associated with a limited vertical depth within the sample, and is one of the collection optical elements.
  • the device forming the aperture can be a slit aperture.
  • the device forming the aperture can be a bundle of optical fibers. Light entering different optical fibers in the bundle of fibers can correspond to light at different vertical depths within the sample.
  • the collection optical elements can collect light from a scan line on the sample with substantially uniform efficiency.
  • the collection optical elements can include a cylindrical lens or a spherical lens.
  • Two or more detectors can be offset from one another with respect to a path for collecting the light from the sample, wherein each of the two or more detectors can be positioned to capture light being emitted from a different vertical depth within the sample.
  • the detector can include a photomultipher detector, a photodiode device, a charge coupled device, or a microchannel plate.
  • Two or more detectors can be provided that receive light from the same region of the sample and detect two or more different characteristics of the sample, such as different polarizations of the light, different frequencies of the light and different lifetimes.
  • Logic can be provided for examining the correlation between the signals obtained from the two or more detectors to identify objects and exclude background noise.
  • the collection of optical data can be limited to regions of the sample known or detected to hold particular objects to be characterized on the sample, and the logic can be implemented in computer software or computer hardware instructions that can be read and executed by a processor operatively connected to the detector.
  • the logic for limiting collection of optical data can limit collection by only recording optical data when an intensity of the collected light is above a certain adjustable threshold value and the optical data meets at least one additional criterion.
  • the logic for limiting collection of optical data can limit collection by only recording optical data during time periods when the beam from the light source is scanned across an area of interest on the sample.
  • the light source can be a continuous wave laser, a pulsed laser, a mode-locked high repetition rate laser, or a Q-switched laser.
  • the continuous wave laser can be a diode laser, a diode-pumped solid state laser, a gas laser, or a tunable solid state laser system and can be modulated in the frequency range of 1 kilohertz to 2 Gigahertz.
  • the pulsed laser can be configured to emit pulses in a frequency range of 1 Hertz - 100 Megahertz with a spacing ranging from 10 nanoseconds to 1 second.
  • the mode- locked laser can have a repetition rate that is higher than or equal to 10 Megahertz.
  • the Q-switched laser can be pulsed at a frequency in the range of 1 Hertz to 1 Megahertz.
  • the light beam emitted from the light source can be intensity modulated in time with a frequency in the range of 1 Hertz to 2 Gigahertz.
  • the scanner can include one or more polygonal mirrors being rotated by a scanning element to scan the light beam across the sample.
  • the scanner can include one or more mirrors being moved by a galvanometer to scan the light beam across the sample.
  • the scanner can be a resonant mirror scanner.
  • the one or more illumination optical elements can include a telecentric lens.
  • the invention provides methods and apparatus, including computer program products, implementing and using techniques for collecting optical data pertaining to one or more characteristics of a sample.
  • the apparatus has a light source, one or more illumination optical elements, a scanner, and one or more collection optical elements.
  • the illumination optical elements direct a light beam from the light source of a first frequency onto the sample.
  • the scanner scans the light beam across the sample.
  • the collection optical elements collect light of a second frequency from the sample and transmit the light to a detector. None of the one or more collection optical elements are included among the one or more illumination optical elements.
  • the invention provides methods and apparatus, including computer program products, implementing and using techniques for collecting optical data pertaining to one or more characteristics of a sample.
  • the apparatus has a light source, one or more illumination optical elements for directing a light beam from the light source onto the sample, a scanner for scanning the light beam across the sample, one or more collection optical elements, a first device that limits detection of light from the sample to light associated with a first vertical depth within the sample, and a second device that limits detection of light from the sample to light associated with a second, different, vertical depth within the sample, where the first and second devices are collection optical elements.
  • Logic can be included for automatically adjusting the vertical position of the sample with respect to the collection optical elements in response to the relative light intensity collected at the first and second devices in order to maintain a consistent vertical position of the sample with respect to the collection optical elements during scanning.
  • At least one of the first device and the second device can be an optical fiber.
  • the first device can include a first row of optical fibers and the second device can include a second row of optical fibers.
  • the one or more detectors can include one or more microchannel plates arranged to separately detect light from the first and second devices.
  • the one or more detectors can include a photomultipher detector, a photodiode device, a microchannel plate, or a charge coupled device.
  • Two or more detectors can be included for receiving light from the sample and detecting two or more different characteristics of the light from the sample.
  • the light beam from the light source can be monochromatic having a first wavelength and wherein one or more of the collection optics and the one or more detectors is tuned to collect light at a second wavelength, which is different from the first wavelength.
  • a third device can be included that limits detection of light from the sample to light associated with a third, different, vertical depth within the sample.
  • the invention can be implemented to include one or more of the following advantages. Improved system and methods for cell and microarray analysis are provided.
  • the use of a scanning light source in combination with improved geometry of the optical collection system, allows for many samples or objects to be illuminated in a single scan. Furthermore, the samples emit radiation in a specific confined region that is amenable to detection with characteristics that allow a higher degree of spatial resolution compared to several existing systems.
  • the use of separate illumination optical components and separate collection optical components reduces the need to separate the illumination light from the fluorescent light emitted by the illuminated sample, and thus provides a simpler and more robust configuration.
  • Using a cylindrical lens, such as a rod lens as one of the collection optical elements allows collection of an entire scan line with substantially uniform efficiency.
  • the polarized nature of the light source can be used to examine reactivity, environment, and/or biological activity of either native material or material that has been tagged with a fluorescent marker.
  • the pulsed or modulated nature of the system allows for time dependent, rapid determination of chemically or photo-induced bioactivity.
  • the timing of the pulses, and the timing of the responses can be used to extract physical information, such as fluorescence lifetimes and polarization relaxation times, as well as chemical or biological information.
  • time-dependent information can be extracted, which can allow for precise mapping into a spatial domain.
  • the optical detection system confines the detection region in such a way that an entire array can be scanned with a precisely located detection region without requiring a conventional autofocus mechanism for the collection optics with the attendant timing requirements.
  • the scanning source focus stays within the confined detection region.
  • the output signal is uniquely suited to analyzing the fluorescence of cells and other objects or features within cells or in solution.
  • the output signal and its characteristic behavior can be analyzed to determine structural, chemical, or biological properties of the object.
  • An image of each object can be spectrally and/or temporally decomposed to discriminate object features by using polarization, fluorescence lifetime, or rotational correlation time as required.
  • An object being imaged in accordance with the present invention can be stimulated into fluorescence, either by autofluorescence, or by binding a molecule or probe, that can be stimulated to fluoresce.
  • Morphological and spectral characteristics of cells and sub-cellular features can be determined by measuring fluorescence signals that may also include time dependent spectral information, which can be used to determine time dependent cellular responses or other information about the cells and their components. Similar measurements can be used to determine nuclear fluorescence intensity, cytoplasm fluorescence intensity, background autofluorescence intensity, fluorescent depolarization intensity, and the ratios of any of these values.
  • the output signal can also be used to monitor the sample's position, and if necessary readjust the position of the sample, such that an optimal amount of light is collected.
  • the output signal can also be used to reduce the data storage requirements, for example, by only storing data when the intensity of the collected fluorescent light is above a certain threshold value.
  • the invention provides methods and apparatus, including computer program products, implementing and using techniques for collecting optical data pertaining to one or more characteristics of a sample.
  • a light beam of a first frequency is scanned onto a sample surface using one or more illumination optical elements.
  • Light of a second frequency is collected from a scan line on the sample surface using one or more collection optical elements. None of the one or more collection optical elements are included among the one or more illumination optical elements.
  • the collected light is transmitted to a detector.
  • Advantageous embodiments can include one or more of the following features.
  • the first frequency and the second frequency can either be the same or can be different.
  • the light can be collected through a device forming an aperture that limits detection of light from the sample to light associated with a limited vertical depth within the sample, wherein the device is one of the collection optical elements.
  • the light can be collected through a slit aperture that limits detection of light from the sample to light associated with a limited vertical depth within the sample.
  • the light can be collected using a bundle of optical fibers, and light that enters different optical fibers in the bundle of optical fibers can correspond to light at different vertical depths within the sample.
  • Light can be collected from a scan line on the sample with substantially uniform efficiency using the one or more optical elements, for example, a cylindrical lens or a spherical lens.
  • the collected light can be transmitted by directing the collected light from the sample to two or more detectors offset from one another with respect to a path for collecting the light, wherein each of the two or more detectors is positioned to capture light being emitted from a different vertical depth.
  • the position of the sample can be adjusted with respect to the collection optical elements in response to light intensity detected at the two or more detectors to maintain a substantially uniform vertical depth from position to position on the sample.
  • the detector can be a photomultipher detector, a photodiode device, a microchannel plate or a charge coupled device.
  • the collected light can be transmitted by directing the collected light from the sample to two or more detectors, and two or more different characteristics of the light from the sample, such as different polarizations, different frequencies of the light, different frequencies of the signal modulation or time-gated regions can be detected.
  • the collection of optical data can be automatically limited to regions of the sample known or detected to hold particular objects to be characterized on the sample. Automatically limiting the collection of optical data can include recording optical data only when an intensity of the collected light is above a certain adjustable threshold value and the optical data meets at least one additional criterion. [0025] Automatically limiting the collection of optical data can include recording optical data only during time periods when the beam from the light source is scanned across an area of interest on the sample.
  • Scanning a light beam can include scanning a light beam from a light source that is one of: a continuous wave laser, a modulated continuous wave laser, a pulsed laser, a mode-locked high repetition rate laser, and a Q-switched laser.
  • the pulsed laser can be configured to emit pulses in a frequency range of 10-100 Megahertz with a spacing ranging from 100 picoseconds to 10 microseconds.
  • the mode-locked laser can have a repetition rate that is higher than or equal to 10 Megahertz.
  • the Q- switched laser can be pulsed at a frequency in the range of 1 Hertz to 1 Megahertz.
  • Scanning can include scanning a light beam from a light source that is intensity modulated in time with a frequency in the range of 1 Hertz to 2 Gigahertz.
  • Scanning can include scanning a light beam with a scanner that includes one or more polygonal mirrors being rotated by a scanning element to scan the light beam across the sample.
  • Scanning can include scanning a light beam with a scanner that includes one or more mirrors being moved by a galvanometer to scan the light beam across the sample.
  • Scanning can include scanning the light beam with a resonant mirror scanner.
  • the one or more illumination optical elements can include a telecentric lens.
  • the invention provides methods and apparatus, including computer program products, implementing and using techniques for collecting optical data pertaining to one or more characteristics of a sample.
  • a light beam of a first frequency is scanned onto a sample surface using one or more illumination optical elements.
  • Light of a second frequency is collected from a scan line on the sample surface using one or more collection optical elements, wherein the light is collected through an aperture that limits detection of light from the sample to light associated with a limited vertical depth within the sample.
  • the collected light is transmitted to a detector.
  • the invention provides methods and apparatus, including computer program products, implementing and using techniques for collecting optical data pertaining to one or more characteristics of a sample.
  • a light beam of a first frequency is scanned onto a sample surface using one or more illumination optical elements.
  • Light is collected from a scan line on the sample surface using one or more collection optical elements.
  • the light is collected through a first device that limits detection of light from the sample to light associated with a first vertical depth within the sample and through a second device that limits detection of light from the sample to light associated with a second, different, vertical depth within the sample.
  • the collected light is transmitted from the first and second devices to one or more detectors.
  • Advantageous embodiments can include one or more of the following features.
  • the vertical position of the sample can be automatically adjusted with respect to the collection optical elements in response to the relative light intensity collected at the first and second devices in order to maintain a consistent vertical position of the sample with respect to the collection optical elements during scanning.
  • At least one of the first device and the second device can be an optical fiber.
  • the first device can include a first row of optical fibers and the second device can include a second row of optical fibers.
  • the one or more detectors can include comprise one or more microchannel plates arranged to separately detect light from the first and second devices. Two or more different characteristics of the light from the sample can be detected.
  • FIG. 1 is a schematic view of an apparatus for collecting optical data in accordance with a first embodiment of the present invention.
  • FIG. 2 is a side elevational view of a first embodiment of a scanner part of the apparatus shown in FIG. 1.
  • FIG. 3 is a side elevational view of a second embodiment of a scanner part of the apparatus shown in FIG. 1.
  • FIG. 4 is an isometric view of the scanner part shown in FIG. 2.
  • FIG. 5 A is an isometric view of the scanner part shown in FIG. 3.
  • FIG. 5B is an isometric view of an apparatus for collecting optical data in accordance with the invention, with an alternative embodiment of the sample array.
  • FIG. 6 is a more detailed schematic view of the detection optics and electronics system of an apparatus for collecting optical data in accordance with the invention.
  • FIG. 7 is a schematic diagram showing a confined field of view for a single detector configuration of the apparatus of FIG. 1.
  • FIG. 8 is a schematic diagram showing a more detailed view of the confined field of view for a single detector configuration of FIG. 7.
  • FIG. 9 is a schematic diagram showing a confined field of view in a stereo configuration of the apparatus of FIG. 1 with multiple detectors.
  • FIG. 10 is a schematic diagram showing multiple confined fields of view for an array of detectors of the apparatus of FIG. 1.
  • FIG. 11 is a schematic diagram showing output signals as a function of time from three individual detectors in a multi-detector configuration of the apparatus of
  • the invention provides an improved apparatus that uses a scanning light source, which can be focused onto an array of samples or objects, with the ability to discriminate against background noise or signal, and makes use of image contrast mechanisms.
  • the apparatus of the invention can be operated in several distinct modes or combinations thereof, depending on what type of sample data needs to be collected. A high-level description of some exemplary modes will first be provided, followed by a more detailed discussion about the parts and geometry of the apparatus.
  • the output signal from the apparatus contains information such as the number of discrete positions in a cell or other object from which the fluorescent light originates, the relative location of the signal sources, and the color (e.g., wavelength or waveband) of the light emitted at each position of the object.
  • the geometry of the illumination optics a relatively large illumination region is created that is confined to a region within the sample volume, thereby eliminating the need to have an apparatus which must adjust the focus of the illumination continuously and an in real time over a plurality or an array of samples.
  • the geometry of the collection optics limits the detection region to a focal volume where the sample is contained and from which the data is collected. In one embodiment, multiple collection arrangements are used with the attendant benefits, which will be described below for a setup with two collection lenses.
  • a plane-polarized laser beam can be propagated through the optical system onto the sample, allowing interrogation of the biological material with polarized light.
  • the emitted light can be separated into its two orthogonal components and analyzed either sequentially in time with a switchable modulator, such as an electrooptic modulator, to allow for detection of the parallel and pe ⁇ endicular components, or simultaneously with multiple collection optics with specified pe ⁇ endicular and parallel polarizing filters.
  • a switchable modulator such as an electrooptic modulator
  • a third mode several laser beams can be propagated through the optical system onto the sample allowing interrogation of the biological material with different wavelengths of light or with the same wavelength at different times.
  • the lasers can be pulsed simultaneously or with a fixed or variable delay between pulses. Delay between pulses allows for measurement of properties of biological materials in an excited state where the first laser pulse causes excitation of the biological moiety and the second or additional laser pulses interrogate that moiety in an excited state.
  • the laser beams can be co-propagated so that they focus on the same sample area during a scan or, alternatively, they can be propagated at some relative angle so that during a scan the laser beams sequentially move over the same sample area.
  • a single modulated laser beam can be propagated through the optical system onto the sample allowing lifetime measurements of the fluorescence in the biological material.
  • several detectors can be used in conjunction with one collection optics arrangement, which creates multiple confinement regions for analysis, the advantages of which will be described in further detail below.
  • several collection optics arrangements can be used to provide improved confinement over a single collection optic with the unique geometry, or can be used to collect emission from the confined region with several characteristics which are uniquely specified to each collecting optics, the advantages which will be described below.
  • an excitation light source (1) emits excitation light (4) to be projected onto a sample (2) that is to be investigated and which rests on a microarray plate.
  • the excitation light source (1) is a laser, such as an Ar or Ar/Kr mixed gas laser with excitation lines of 488, 514, 568 and 647 nm.
  • a continuous wave (CW) laser such as the Compass 315 M laser from Spectraphysics Inc. of Mountain View, CA, is used as an excitation source.
  • the wavelength of the excitation light can be either within the visible range (i.e., 400-700 nm), or outside the visible range. For excitation wavelengths below 400 nm photochemical reaction rates, such as those due to photobleaching, tend to be substantial.
  • the output from the laser (1) can be modulated and give information about the time dependent response of fluorescence signals by using a frequency modulation detection scheme.
  • a pulsed laser with laser pulses of approximately 12 ps FWHM (Full Width at Half Max) with a spacing of approximately 12 ns is used as the excitation light source (1).
  • the average power of the laser (1) at the sample (2) is typically in the range lmW - 1W.
  • the spacing of 12 ns is convenient for fluorescent lifetime detection, but can be varied as necessary, for example, by varying the cavity length of the laser (1).
  • Common to both embodiments is the use of time-resolved imaging as a contrast producing agent. This has been developed significantly in the field of fluorescence microscopy and has been described in detail by Marriott, Clegg, Arndt-Jovin, and Jovin, 1991, Biophys. J. 60:1374-1387; Verveer, Squire, and Bastiaens, 2000, Biophys. J. 78:2127-2137; Buehler, Dong, So, French, and Gratton, 2000, Biophys.
  • the excitation light (4) passes through one or more illumination optical elements to the sample (2).
  • the illumination optical elements include an electro-optic modulator (8), a set of beam-shaping lenses (3), a scanning device (5), and a multi-element lens (9).
  • the electro-optic modulator (8) can be used to polarization modulate the excitation light (4), if required by the investigation that is to be carried out on the sample (2).
  • the set of beam-shaping lenses (3) expands the laser beam in order to match the input aperture of the scanning lens and provide the desired illumination region size at the sample (2).
  • the scanning device (5) moves the expanded laser beam back and forth in a line-scan over the sample (2) after the beam has been focused by the multi-element lens (9).
  • the scanning device (5) can be an electromechanical device coupled to an optic element, such as a mirror driven by a galvanometer.
  • the scanning device (5) uses a polygon with multiple reflective surfaces to scan the laser beam across the sample (2).
  • the multi-element lens (9) is designed to focus the laser light at the operating wavelength of the laser (1).
  • the multi-element lens (9) can, for example, be a microscope objective designed for the operating wavelength or a specially designed scanning lens, such as a telecentric lens, that has appropriate parameters to achieve a flat focal plane, for example, with a long working distance and low first and second order aberrations, thus producing the same spot size and shape over a wide range of positions (such as a scan line).
  • the telecentric lens is particularly useful for covering a large field of view.
  • the beam (10) is focused onto a region of the sample (2) to be imaged.
  • the focal region is located above, for example, a base of a microarray plate.
  • the sample (2) can be objects to be interrogated by fluorescence, such as cells attached to the bottom of a microwell of the microarray plate.
  • the fluorescent light emitted by the sample (2) is collected by one or more collection optical elements (19).
  • the collection optical elements (19) is a rod lens, designed to capture the entire range of sweep of the beam (10) over one dimension of the base (11) of the sample array.
  • the collection optical elements (19) can also include other types of lenses, or an aggregate of lenses, as would be determined by the specific information required from the emission. In some embodiments, multiple setups of collection optical elements (19) can be used to improve collection efficiency.
  • the light collected by the collection optical elements (19) is transmitted to a detector (21) located at a convenient distance from the collection optical elements (19).
  • the transmission of the fluorescent light can be accomplished by, for example, an optical fiber or a bundle of optical fibers (20).
  • the detector (21) is a detector with high gain, such as a photomultipher tube, which produces an electrical output signal.
  • the electrical output signal is further processed by a data acquisition system (14), which performs operations such as optimization of the gain and the signal to noise ratio (S/N), by making use of signal enhancing, averaging, or integrating detection systems.
  • FIG. 2 shows a side elevational view of the scanning portion of a first embodiment of an apparatus in accordance with the invention.
  • FIG. 4 shows an isometric view of the scanning portion of the same embodiment of the apparatus.
  • the scanning device (5) is a mirror (6) driven by a galvanometer. By moving the mirror (6) back and forth using the galvanometer, the excitation light (10) from the laser (1) can be swept across the sample (2).
  • FIGs. 3, 5A and 5B show similar views of a second embodiment of an apparatus in accordance with the invention, where the scanning device (5) instead is a polygon (7) with multiple reflective surfaces. In this embodiment the laser beam (10) is swept over a region of the sample (2) by rotating the polygon (7).
  • the scanning device (5) is a resonant scanning device, such as a mirror mounted on a torsion bar with electromagnets causing the mirror to move back and forth.
  • the beam velocity across the sample (2) is thus a result of the rotation speed of the polygon (7) or the sweep velocity of the galvanometer and the resonant scanning device, respectively.
  • the galvanometer is less expensive than the polygon mirror, but operates at a smaller angle and at a lower frequency, which causes a slower scanning speed.
  • the resonant scanning device is cheaper than both the galvanometer and the rotating mirror and operates at larger angles, but only operates at a predetermined frequency.
  • the beam motion at the focal plane in the sample (2) is typically 1-10 mm/ms, but can be as fast as 10-1000 mm/ms, depending on the sweep velocity of the mirror (6), or the rotation speed of the polygon (7).
  • the polygon (7) is typically rotated at rotation speeds from 0.5 kHz to 20 kHz.
  • the multi-element lens (9) that receives the laser light (4) is designed to focus the laser light at the operating wavelength of the laser (1).
  • the multi-element lens (9) focuses the laser light (4) close to the diffraction limit of the multi-element lens (9), which is typically in the range of 5 - 20 microns, but can be as small or large as 1-200 microns.
  • the sample or sample array (2) is arranged to accept the focused, beam at, or just above, the base (11) of the sample (2).
  • the length of the scan line across the sample array (2) can be varied and is typically in the range 5 mm to 100 mm.
  • the scan light (10) can interrogate for example, a 96-well plate in less than one minute at 5 micron resolution.
  • an optical element (12) such as a mirror, is provided approximately half way between the scan lens and the sample to intercept and reflect a section of the incident light (10) onto a detector (13).
  • the reflector (12) is located about 1-2 cm from the scan lens.
  • the detector (13) is used to detect the location of the start of scan, in order to trigger the data acquisition system (14), which will be described in further detail below.
  • the detector (13) can, for example, be a photodiode or equivalent component that can sense the incoming light (10) reflected from the reflector (12) and provide an electrical signal to the data acquisition system (14).
  • FIG. 7 shows an enlarged view of the sample (2), how incoming light (10) illuminates the sample (2), and a source region (17) from which the fluorescent light is collected in a single detector embodiment of the apparatus of FIG. 1.
  • the sample (2) is located on a base (11) with a series of optical elements (16) that allow the laser light (10) to be transmitted through to the sample contained in the array.
  • the array can, for example, be a microarray plate containing wells with solutions or samples adhered to the bottom of the wells.
  • the focal plane location is near the inner side of optical elements (16) and defines the region of highest light flux, thereby defining a region of highest emitted light source.
  • the region's volume size depends on the multielement lens (9) configuration and the depth of the interrogated sample (2) located above the base (11).
  • the defined volume of a source region (17), which actually gives rise to the fluorescent signal, additionally depends on the configuration of the collection optical elements (19), as will now be discussed.
  • the geometry of the collection optical elements (19) is such that the collection region is confined to the region of the field of view for the detector (21).
  • the fluorescent signal intensity is confined to a source region (17) formed by the intersection of the excitation source's focal region and the image of the detector (21) inside this region, as shown in FIG 7.
  • the source region is located within a limited vertical depth of the sample, that is, at a limited distance range above the base (11) upon which the sample (2) rests.
  • the collection region is fixed or confined by the collection optical elements (19) configuration so as to not be out of the focal plane of the system. Yet another advantage is that signal discrimination from background fluorescence in the sample well is much higher than that obtained by a parallel collection system without eliminating or filtering the fluorescent signal. [0062]
  • the emitted fluorescent light from the source region (17) is transmitted to the collection optical elements (19) along the collection path (18).
  • the collection path is fixed or confined by the collection optical elements (19) configuration so as to not be out of the focal plane of the system. Yet another advantage is that signal discrimination from background fluorescence in the sample well is much higher than that obtained by a parallel collection system without eliminating or filtering the fluorescent signal.
  • the collection path can extend through the well in the sample array to a location on the opposite side of the sample array, as shown in FIG. 1, for example.
  • the collection optical elements (19) are configured to collect and focus the light emitted from the source region, as was described above.
  • FIGs. 4, 5A and 5B There are several ways to configure the collection optical elements (19) that allow the scanning of a large array, such as a microarray plate.
  • One geometry is shown in FIGs. 4, 5A and 5B.
  • the collection optical elements are shown in FIGs. 4, 5A and 5B.
  • the collection optical elements (19) can include other types of lenses or lens combinations, as would be determined by the specific information required from the fluorescent emission. As a result of light collimation by a single collection lens (19) as shown in FIGs. 4, 5A and 5B, all light emitted from a position on the array cell or microarray plate can be imaged, and collected with high efficiency.
  • another embodiment of the collection optical elements (19) includes an optical transmission filter (23) and a slit aperture (26).
  • the transmission filter (23) Before passing the fluorescent light collected by the rod lens (19) to the detector (21), the light is appropriately filtered by the transmission filter (23), which is designed to pass the fluorescence emission.
  • the transmission filter (23) which is designed to pass the fluorescence emission.
  • several filters can be chosen to minimize the amount of laser light to be detected by the detector (21).
  • the optical filter (23) is chosen to optimize the collection of information within the spectral region of light emitted by the source region (17).
  • the laser light is between 400 and 500 nm in wavelength
  • the emitted fluorescence is in the region above 500 nm
  • the optical filter (23) is a 500nm long pass filter located behind the rod lens (19).
  • Many other configurations can be envisioned by people skilled in the art, depending on the wavelengths of the incident and the emitted light, and the filters chosen.
  • the slit aperture's (26) opening is located directly in front of the entrance to the detector (21) or optical fiber (20) coupled to the detector (20).
  • the light that is emitted from the center of the source region (17) is collected by the rod lens (19) and passes through the center of the slit aperture (26).
  • light that is emitted from regions at a different depth of the sample, such as from the edge of the source region (17) will be imaged by the rod lens (19) outside the slit aperture's (26) opening, and will thus not be collected.
  • the advantage of further confining the focal region is that an improved spatial resolution will result, as well as further discrimination of background fluorescence outside of the region.
  • an aperture size of 250 microns results in approximately a 400 micron detection region.
  • combinations are also possible in which there is only an optical transmission filter (23) or slit aperture (26), but not both.
  • two or more collection optics arrangements (19a, 19b) are provided.
  • the focal field for the two lenses can have improved confinement over the single field generated by one lens and the focusing source shown and discussed above with respect to FIG. 8.
  • the improvement is schematically represented in FIG. 9 by the intersection (22) of the focal planes for the respective collection optics arrangements (19a, 19b), corresponding to the main object planes of the lenses (19a, 19b).
  • the setup of FIG. 9 with two sets of collection optics (19a, 19b) can also be used for simultaneous collection of orthogonal components of emission from a polarized excitation source.
  • a first polarizing filter (23a) can be used to pass only light of a first polarization to a first detector (21a), and a second polarizing filter (23b) can be used to pass only light of a second, orthogonal, polarization to a second detector (21b).
  • the correlation of the signals collected in this configuration, detection in the detection system, and subsequent manipulation of the stored signal give rise to information not available to a single detector, with attendant improvement in signal.
  • the information derived from this apparatus is steady-state anisotropy.
  • Time-resolved anisotropy of the emissions signal can give dynamical and/or structural information on biomolecules and their environment. It is important that any polarization filtering is performed before the collected light enters any optical fibers, since the optical fibers distort the polarization information and light that is output from an optical fiber does not have identical polarization components to the light that was input to the optical fiber at the other end.
  • the detector (21) can be a detector with high gain, such as a photomultipher tube (PMT).
  • PMT photomultipher tube
  • Other examples of detectors are photodiodes, various types of charge coupled devices (CCDs), or microchannel plates.
  • the detector (21) does not have to be physically located adjacent to the collection optical elements (19), but the light can be transmitted from the collection optical elements (19) to the detector (21) through a fiber array (20).
  • multiple detectors (21a-21c) are arranged adjacent to each other in order to collect the signal from the collection optical elements (19).
  • the individual detectors (21a-21c) each have their own confined field of view, with the attendant advantages associated with the confined focal region as described above for one detector.
  • the multiple detectors (21a-21c) do not have to be physically located adjacent to the collection optical elements (19), but the light can be transmitted from the collection optical elements (19) to each of the detectors (21a-21c) through a fiber array (20), or relay lens system for each detector.
  • This multi-detector arrangement has additional advantages, such as the ability to simultaneously detect signal at multiple locations, such as at different depths, within the source region (17) and to assign these signals to spatial locations within the sample (2).
  • the multiple detectors (21a-21c) can be configured with optical filters (not shown in FIG. 10), and used to collect fluorescent emission from different spectral regions.
  • the multiple detectors (21a- 21c) can be configured to detect orthogonal polarization signals, as described above, allowing for simultaneous detection of the anisotropy of the fluorescent signal.
  • the detectors (21a-21c) can also be used to correct the sample position based on the recorded signals, as can be seen in FIG. 11. Assume, for example, that it is desired to keep the sample (2) aligned with the collection optics, so that most of the signal is received by the middle detector (21b). Since each detector (21a-21c) is associated with a different depth, it can be expected that the middle detector (21b) should have a signal that is higher than the outer detectors (21a, 21c). As can be seen in FIG.
  • This technique can be used to move the sample (2) not only in the vertical direction, but also in the horizontal direction, depending on the detector arrangement. If multiple detector arrangements are used, such as in three orthogonal directions, complete control over the sample positioning can be achieved in all spatial directions. Since movement within a horizontal plane can occur with two degrees of freedom, it is necessary to have two sets of detectors that preferably are oriented pe ⁇ endicular to each other within the horizontal plane. With this detector arrangement, a horizontal translation of the sample will result in an increased signal in one or both detector sets, and the movement can be unambiguously identified.
  • the apparatus also contains logic, such as a data acquisition system (14), a data processing and storage system (24), and a controller (15), which work in conjunction with the above-described optical and mechanical components of the apparatus to provide adequate control capabilities for the various types of investigations that can be carried out with the apparatus.
  • the signal from the detector (21) is enhanced by the data acquisition system (14), and then stored into the data processing and storage system (24).
  • the data processing and storage system (24) contains a fast A/D converter, or accepts digital information from the data acquisition system (14) directly.
  • the data processing and storage system (24) can, for example, be a digitizing storage oscilloscope, or a computer with instructions encoded in software for collecting and storing the detected or enhanced emission signal.
  • the signal can be labeled using a triggering event in time, and can be co- located with a spatial position of the fluorescing object within a well of a microarray, or with the macro location of the well in the microarray plate.
  • the software logic in the data processing and storage system (24) can contain instructions for deriving one or more object characteristics from the emission signal, such as total intensity, average intensity, peak intensity, size, Gaussian or other waveform fit, or other such characteristics as may be found useful to those skilled in the art.
  • the trigger signal can be modified by the controller (15) as needed to configure a delay, a blanking signal, a duty cycle, or provide a means by which the trigger circuit of a boxcar averager, for example, can be activated.
  • Two triggering events at the start and end of a scan can be used to measure the total scan time and correct for scan jitter. This also enables bidirectional scanning. There are many permutations for using this data processing and data storage system (24) that are not described here, but which are useful to those skilled in the art.
  • the data processing and storage system (24) can be set up such that data is only collected and saved when a relevant part of the sample (2), such as a cell, is illuminated. In one embodiment, this is accomplished by setting a threshold value in the data processing and storage system (24), and saving data only when the intensity of the collected fluorescent light exceeds the threshold value for a certain period of time, or whenever some other pre-determined criterion is satisfied. In another embodiment, the data processing and storage system (24) only saves data during certain time intervals, such as when the illuminating beam (10) illuminates a well or a location in a microarray.
  • the apparatus allows for measurement of successive laser pulses, as a result of modulating the laser light, over the same spatial location of the scan region and then subsequently analyzing the fluorescent signal measured by the detector (21) to determine a time-dependent response of the sample within the scanned region.
  • the response can include one or more characteristics of the sample, such as molecular interactions, protein-protein interaction, binding kinetics, drug/target interactions, cell apoptosis, and so on.
  • the timing and response to time dependent perturbations, such as the excitation pulse form important aspects of this invention.
  • the detector (21) can be arranged to collect information stored in the incident light as well as the emitted light, such as the polarization of the light.
  • the light source (1) is polarized, the incident polarization is determined, and the fluorescent response emitted by the sample (2) is analyzed for its polarization components, or anisotropy.
  • the polarization of the incident light and/or the fluorescent light can be modulated, for example, by the electrooptic device (8).
  • the timing of the modulation of the polarized signals is controlled by the controller (15) with respect to the timing of the scans, so that quick, successive scans with orthogonal polarization can be performed and so that dynamical information from the fluorescent polarization can be extracted.
  • the intensity of the incident light can be modulated to collect time-dependent information from the sample.
  • the sample (2) can be placed on a moveable platform (25) that can be used to position the sample (2).
  • the platform can handle a microarray plate containing 96- sample wells, or a 3456-well plate for addressing very large arrays of tests and samples.
  • a raster scan, or focused line of light (10) is provided to the sample (2) and the emission is collected by the collection optical elements (19) in such a way the arrays can be addressed in a parallel fashion.
  • the parallel addressable nature of the invention allows for very high throughput scanning and data collection, which is useful for example, for interrogating and screening therapeutic effects of chemicals on biomaterials as described above.
  • the platform (25) can be configured to move with a precision that is either less than or on the order of the optical resolution of the multi-element lens (9), such that the motion of the platform (25) gives rise to high-resolution images of the sample (2).
  • the scanned beam (10) is swept across the sample (2) in one dimension and the sample array is moved in a pe ⁇ endicular direction to the sweep by the platform (25), whereby the movement is timed such that the beam makes one or more complete excursions, and the emission signal from the detector (21) derived from one or more complete sweeps is collected and summed or manipulated by the data acquisition system (14) and the data processing and storage system (24).
  • the platform (25) motion is pe ⁇ endicular to the motion of the scan (10), such that a two-dimensional image of the sample (2) can be reconstructed using the instructions encoded in the data processing and storage system (24).
  • the focus location of the multi-element lens (9) in the source region (17) can provide spatial information in the direction pe ⁇ endicular to the plane defined by the scan (10) and platform (25) motion, resulting in a reconstructed 3 -dimensional image.
  • the time domain information reconstructed by the data acquisition system (14) and the data processing and storage system (24) can be used to construct image spatial locations, which can give rise to information on the objects in sample array, such as events that occur as a result of the light probe.
  • the information may result from, for example, non-light-induced drug or responses at the cellular or subcellular level.
  • the apparatus may perform the scanning function by moving the sample (2) only, instead of using a scanning device (5) to move the beam (4) from the light source (1) across the sample.
  • the invention has been described above with regards to fluorescent light, but the same principles can be applied to the collection of phosphorescent light, which may be useful for investigations of certain samples.
  • the invention can also be used to perform measurements of chemiluminescence and resonant energy transfers. Accordingly, other embodiments are within the scope of the following claims.

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Abstract

L'invention porte sur des procédés et des appareils incluant des programmes informatiques et mettant en oeuvre et utilisant des techniques de recueil de données optiques relatives à une ou plusieurs caractéristiques d'un échantillon. L'appareil comporte une source lumineuse, un ou plusieurs éléments optiques d'éclairage, un dispositif de balayage, un ou plusieurs éléments optiques de recueil, et un dispositif présentant une ouverture limitant la détection de la lumière provenant d'un échantillon. Les éléments optiques d'éclairage dirigent sur l'échantillon le faisceau lumineux provenant de la source lumineuse. Les éléments optiques de recueil collectent la lumière provenant de l'échantillon et la transmettent à un détecteur. Aucun des éléments optiques de recueil ne figure parmi les éléments optiques d'éclairage. L'élément présentant l'ouverture, et qui restreint la détection de la lumière provenant de l'échantillon à sa partie associée à une profondeur verticale limitée de l'échantillon, est l'un des éléments optiques de recueil.
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EP1660870A2 (fr) 2006-05-31
EP2259050A2 (fr) 2010-12-08
US7812956B2 (en) 2010-10-12
US20050046849A1 (en) 2005-03-03
US20050046848A1 (en) 2005-03-03
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